The promiscuous development of an unconventional Qa1b-restricted T cell population

MHC-E restricted CD8 T cells show promise in vaccine settings, but their development and specificity remain poorly understood. Here we focus on a CD8 T cell population reactive to a self-peptide (FL9) bound to mouse MHC-E (Qa-1b) that is presented in response to loss of the MHC I processing enzyme ERAAP, termed QFL T cells. We find that mature QFL thymocytes are predominantly CD8αβ+CD4-, show signs of agonist selection, and give rise to both CD8αα and CD8αβ intraepithelial lymphocytes (IEL), as well as memory phenotype CD8αβ T cells. QFL T cells require the MHC I subunit β-2 microglobulin (β2m), but do not require Qa1b or classical MHC I for positive selection. However, QFL thymocytes do require Qa1b for agonist selection and full functionality. Our data highlight the relaxed requirements for positive selection of an MHC-E restricted T cell population and suggest a CD8αβ+CD4- pathway for development of CD8αα IELs.


Introduction
Thymic development of conventional CD8 T cells requires low affinity recognition of self-peptides bound to MHC I molecules expressed by cortical thymic epithelial cells and gives rise to naïve circulating CD8 T cells.Conventional CD8 T cells recognize peptides bound to classical MHC I (called MHC Ia) molecules, in contrast to unconventional T cell populations that recognize a diverse set of non-classical MHC I (called MHC Ib) (1,2).MHC Ib molecules are structurally homologous to MHC Ia, and often associate with b2m, but are generally non-polymorphic, and can bind peptides or non-peptidic ligands (3).The two most prominent and well-studied examples of unconventional abTCR-expressing T cells are mucosal associated invariant T cells (MAIT cells), that recognize vitamin B metabolites presented by MR1, and invariant natural killer T cells (iNKT cells), that recognize lipid metabolites presented by CD1d.MAIT cells and iNKT cells, like conventional T cells, require their cognate MHC ligand to develop in the thymus (4,5).However, unlike conventional T cells, which undergo positive selection by weak TCR signals, MAIT and iNKT cells undergo "agonist selection", an alternative thymic selection process in which strong TCR signals drive alternative differentiation programs instead of negative selection (6).In addition, MAIT and iNKT cells recognize self-ligands presented by thymic antigen presenting cells (APCs) of hematopoietic, rather than epithelial, origin (4,5).The thymic development of T cells restricted to other MHC Ib molecules remains understudied (7)(8)(9)(10)(11)(12).
While MHC Ib restricted T cells are relatively rare in circulation, they contribute substantially to the intraepithelial lymphocyte (IEL) compartment of the small intestine (13-15).abTCR+ IEL are generally classified as either induced or natural IELs, which differ in their specificity and developmental pathways.Induced IELs, which express the CD8ab heterodimer, are specific for classical MHC Ia molecules and are derived from conventional CD8 T cells following antigen encounter in the periphery (16,17).On the other hand, natural IELs, which predominantly express the CD8aa homodimer, can recognize a variety of different MHC ligands and are programmed for an IEL fate by strong recogniton of self ligands in the thymus (18)(19)(20).Studies of natural IEL development have largely focused on populations of abTCR+CD4-CD8-(double negative or DN) thymocytes, which can give rise to CD8aa IEL upon transfer into T cell deficient mice (21)(22)(23)(24)(25).However, it is unclear whether all natural IEL develop via an abTCR+DN stage.Moreover, while it is known that many natural IEL require b2m, but not MHC Ia molecules, for their development (13-15, 18, 19), the specifity of IEL for particular MHC Ib molecules remains largely unknown.As a result, no studies to date have focused on the thymic development of IELs specific for defined MHC Ib molecules.
The MHC Ib molecule MHC-E (called Qa1 in mouse) is best known for its role in regulating NK cell responses; however, recent attention has focused on its function as a restricting MHC molecule for CD8 T cells (26,27).In healthy cells, MHC-E molecules predominantly display a self-peptide derived from an MHC Ia leader peptide (called QDM peptide in mouse), which serves as a ligand for NK receptors and provides an inhibitory signal to NK cells and activated T cells (28,29).However, under conditions of impaired MHC Ia presentation, such as deficiency in ERAAP (endoplasmic reticulum aminopeptidase associated with antigen processing) or TAP (transporter associated with antigen processing), Qa1 b is loaded with an alternative set of peptides that can be recognized by CD8 T cells (10,(30)(31)(32).MHC-E restricted T cells responsive to TAP and ERAAP deficient cells have been proposed to play a role in monitoring defects in MHC Ia presentation induced by viral infection (33), transformation, or stress (31,32).In addition, pathogen-specific MHC-E restricted CD8 T cells can be activated upon infection with a variety of viruses and bacteria (34)(35)(36)(37)(38). Recent studies of a CMVvectored anti-HIV vaccine showed that MHC-E restricted CD8 T cells can produce responses that are extremely broad, with an unusually large proportion of the potential epitopes being targeted for recognition, and which provide strong immune protection (39,40).Altogether, the ability of MHC-E restricted T cells to respond broadly to both microbial antigens and abnormal self, suggests an unusual mode of T cell recognition with significant therapeutic potential.However, our limited understanding of the specificity and development of MHC-E restricted CD8 T cells hampers our ability to harness these responses for therapeutic purposes.Perhaps the best characterized example of an MHC-E restricted CD8 T cell response is QFL T cells, which recognize Qa1 b loaded with a self-peptide FYAEATPML (FL9) derived from Fam49a/b proteins (31).QFL T cells were discovered as part of the mouse T cell response upon immunization of wild type mice with ERAAP deficient splenocytes.Interestingly, QFL T cells display hybrid characteristics of both conventional and unconventional T cells.Like conventional MHC Ia-restricted T cells, QFL T cells are found in the spleen and express the CD8ab heterodimer.However, reminiscent of MAIT and iNKT cells, the majority use a semi-invariant TCR with a fixed TCRa and limited TCRb usage (41).Splenic QFL T cells display an antigen experienced phenotype in wild type, unimmunized mice, reminiscent of conventional CD8 T cells that acquire a memory phenotype following homeostatic proliferation to self, termed "memory phenotype" or "virtual memory" T cells (42,43).While QFL T cells can be detected using FL9-Qa1 b tetramers (called QFL tetramers) in wild type and Qa1 b deficient mice (31), their development in the thymus, and their contribution to the IEL compartment have not yet been examined.
Here we use both QFL tetramers and mice expressing rearranged QFL-specific abTCR transgenes to probe the development of QFL T cells in wild type and MHC I deficient mice.QFL T cells can be readily detected in the spleen, thymus, and IEL compartment, with QFL T cells in the IEL compartment comprised of both CD8aa and CD8ab phenotypes.Our data indicate that Qa1 b expression, predominantly by hematopoietic cells, drives the agonist selection of QFL T cells in the thymus, leading to mature CD8+CD4-thymocytes that exhibit signs of strong TCR signals.However, QFL T cells also recognize an alternative MHC I ligand, which can allow for positive selection of QFL CD8SP thymocytes with a more conventional phenotype in the absence of Qa1 b .Our data highlight the promiscuous recognition and development of QFL T cells, confirm their hybrid conventional/unconventional characteristics, and suggest an alternative pathway for the development of natural IELs.

Characterization of QFL T cells in wild type and TCR transgenic mice
To investigate the development of QFL specific T cells, we used tetramer enrichment of lymphocytes using Qa1 b tetramers loaded with the FL9 peptide (31) (hereafter called QFL tetramers).To increase the specificity of detection, we co-stained using both the QFL tetramer and an antibody specific for Va3.2, which recognizes the invariant TCRa chain used by the majority of QFL T cells (Supplementary Figures 1A, B; Figure 1A) (41).In this study, we focused on QFL tetramer + and Va3.2 + cells, hereafter called QFL T cells.The majority of QFL T cells in the thymus, spleen and small intestine (SI) intraepithelial lymphocyte (IEL) compartment of wild type mice were CD8a + CD4-(Figure 1A).Interestingly, while mature QFL T cells in thymus and spleen predominantly expressed the CD8ab heterodimer, QFL T cells in the IEL compartment were a mixture of cells expressing CD8aa or CD8ab (Supplementary Figures 1C-E).As previously reported (31), QFL T cells were relatively abundant in the spleen of wild type mice (~1300/spleen/ mouse or 1/6000 CD8 T cells, Figures 1B, C).For comparison, a study of conventional CD8 T cell frequencies reported a range of 1/30,000 to 1/160,000 (42,44).Additionally, a substantial number of QFL T cells were identified in the thymus and IEL compartment of the small intestine, with an average of 240 QFL T cells and 1,200 QFL T cells respectively (Figure 1B).The frequency of QFL T cells out of mature CD8 T cells was higher in the SI IEL compared to the thymus and spleen (Figure 1C), suggesting that they undergo selective recruitment and/or expansion in this compartment.
As a complimentary method to characterize QFL T cells, we developed a TCR transgenic mouse that expresses the semiinvariant QFL TCR a-and b-chain (Va3.2Ja21,Vb1Db1Jb2-7) used by a predominant clone (41), henceforth referred to as QFLTg.As expected, mature (CD24-) thymocytes expressing the QFL TCR were predominantly CD8 single positive (SP) (Supplementary Figure 1A).DP thymocytes from transgenic mice expressed relatively low levels of the QFL TCR, while CD4SP thymocytes expressed moderately high levels of the TCR but remained mostly immature (CD24+) (Supplementary Figure 2A).To confirm that selection of the QFL CD8SP T cells in these mice was driven by the transgene encoded TCR, we examined expression of Va2, which is expressed on ~10% of T cells in wild type mice and serves as a representative of endogenous TCRa expression.As expected, mature CD8SP in thymus and spleen of QFLTg mice had reduced expression of Va2, whereas mature CD4SP has levels of Va2 similar to wild type, confirming that CD8SP are mostly selected using the QFL TCR, whereas CD4SP are selected using endogenous TCRs (Supplementary Figures 2B, C).Importantly, the population defined as QFL CD8SP in this study (gated: QFL tetramer+Va3.2+CD8+CD4-) was almost completely devoid of endogenous TCRa expression.These data validate the efficacy of our TCR transgenic system and confirms that QFL CD8SP in transgenic mice are selected by the transgene-encoded TCR.
We also used thymocytes and splenocytes from QFLTg mice to confirm the specificity of the QFL TCR for the FL9-Qa1 b complex.As expected, the majority of QFL tetramer+ thymocytes and splenocytes did not stain with tetramers in which Qa1 b was loaded with the predominant QDM peptide (Supplementary Figure 2D).The small population of QFL tetramer+ cells that did stain with the QDM tetramer also expressed the QDM/Qa1 b receptor NKG2A, an NK receptor that is also expressed by activated T cells.The co-staining of QDM tetramer and NKG2A suggests TCR independent binding of QDM tetramer on QFL T cells and serves as a positive control for QDM tetramer staining.These data confirm the specificity of the QFL TCR for the FL9/ Qa1 b complex.
Previous studies showed that QFL T cells respond to a selfpeptide presented by Qa1 b (31).In addition, splenic QFL T cells from wild type mice display an antigen experienced phenotype, suggesting that they may receive strong TCR signals during their development in the thymus.To test this notion, we examined expression of CD5, a marker which positively correlates with self-reactivity (45)(46)(47)(48).As predicted, CD5 is elevated in QFL CD8SP thymocytes from QFLTg and non-transgenic mice compared to conventional CD8SP T cells (Figure 1D).In addition, QFL CD8SP thymocytes from nontransgenic mice showed slight but detectable downregulation of CD8b compared to conventional CD8SP T cells, whereas QFL CD8SP thymocytes from QFLTg mice showed more pronounced CD8b downregulation (Figure 1E).This modulation of CD8b expression has been associated with thymocyte self-reactivity and agonist selection (49,50).QFL CD8SP thymocytes from QFLTg mice also showed elevated levels of several markers associated with agonist selection, such as the transcription factors PLZF (51,52) and Tbet (Supplementary Figure 3).On the other hand, splenic, but not thymic, QFL CD8SP T cells express elevated levels of CD44, a marker associated with antigen experience (Figure 1F; Supplementary Figures 3A-C).In addition, QFL CD8SP thymocytes from QFLTg mice did not show detectable upregulation of PD1 or a4b7, markers that are expressed by a subset of thymic IEL precursors (Supplementary Figures 3A-C) (22).Taken together these data suggest that QFL T cells experience relatively strong TCR stimulation and undergo agonist selection during their development in the thymus.

QFL T cell development in absence of Qa1 b or classical MHC I
Some agonist selected T cell populations, such as regulatory T cells require a separate positive selection interaction prior to undergoing agonist selection (6).Previous reports that QFL T cells are detectable in the spleen of mice lacking Qa1 b , but undetectable in mice lacking b2m (31), a subunit of MHC I which is required for proper folding and surface expression of both classical MHC Ia and Qa1 (53), suggested the possibility that QFL T cells require positive selection on classical MHC Ia.To test this hypothesis, we generated K b D b KO mice and compared the number of QFL T cells in the thymus and spleen to that of WT and Qa1 b KO mice (Figure 2A; Supplemental Figures 4A, B).The QFL CD8SP thymocytes were slightly reduced in K b D b KO and Qa1 b KO relative to wild type mice, but were undetectable in b2mKO mice (Figure 2A).Similar results were obtained with QFLTg mice, with substantial numbers QFL thymocytes and splenocytes found in the absence of Qa1 b or K b D b , but not in the absence of b2m (Figure 2B).Importantly, the QFL CD8SP thymocytes and splenocytes from QFLTg, QFLTgQa1 b KO mice have negligible expression of endogenous Va2 (Supplemental Figures 2B, C), confirming that these cells were positively selected using the QFL TCR.Classical MHC Ia D b is the source for the QDM peptide that is bound to Qa1 b in ERAAP sufficient cells, raising the possibility that loss of D b could indirectly impact QFL T cell development by altering the peptides displayed on Qa1 b .However, QFL CD8SP thymocytes and splenocytes were found in similar numbers in K b KO, D b KO, and K b D b KO mice, arguing against this possibility (Supplementary Figure 4C).Altogether, these data suggest that neither classical MHC Ia, nor Qa1 b , are required for QFL T cell positive selection, although both may contribute to the efficiency of the process.Interestingly, CD8SP T cells in spleens of K b D b KO mice exhibit a higher frequency of Va3.2 + cells compared to WT or Qa1 b KO mice (Supplementary Figure 4D), indicating that this V segment is preferentially used by T cells reactive to MHC Ib molecules.Because Qa1 b presents agonist FL9 peptide to QFL T cells, we hypothesized that expression of Qa1 b might lead to agonist and negative selection of QFL thymocytes.In support of this, DP thymocytes in QFLTg (Qa1 b sufficient) mice exhibit reduced cellularity and a "DP lo " phenotype associated with strong TCR signals (19,(54)(55)(56) whereas QFLTg Qa1 b KO mice express normal levels of CD4 and CD8a (Figure 2C).In addition, QFL CD8SP thymocytes from Qa1 b sufficient, but not Qa1 b KO mice, displayed CD8b downregulation, PLZF expression, and elevated CD5 expression compared to conventional mature CD8SP thymocytes (Figures 2D-F).In contrast, in the absence of classical MHC I (K b D b KO mice) QFL CD8SP T cells showed strong downregulation of CD8b expression, maintained PLZF expression and showed a slight reduction in CD5 expression compared to WT mice (Figures 2D-F).In the periphery, QFL T cells lost their antigen experienced phenotype in absence of Qa1 b , but not in absence of classical MHC I (Supplemental Figure 4E).These data suggest that Qa1 b is required for agonist selection of QFL T cells, although positive selection of QFL T cells can be driven by an alternative MHC 1 molecule.

QFL T cells recognize an alternative ligand on Qa1 b KO APCs
To further explore the ligand-specificity of the QFL TCR we took advantage of the observation that MHC-naïve DP thymocytes are highly sensitive to in vitro TCR stimulation (57,58).We examined expression of activation markers on preselection QFLTg (preQFLTg) thymocytes from a b2mKO background after co-culture with bone marrow derived dendritic cells (BMDC) isolated from mice lacking either Qa1 b , K b D b or b2m.PreQFLTg thymocytes showed stronger upregulation of the activation markers CD69 and CD5 upon 24-hour co-culture with WT and K b D b KO, compared to Qa1 b KO, BMDC (Figure 2G; Supplementary Figure 5A).This is consistent with the hypothesis that Qa1 b is the primary MHC ligand for the QFL TCR.Interestingly, preQFLTg thymocytes showed a modest activation when co-cultured with Qa1 b KO BMDCs; this activation was significantly more compared to co-culture with b2mKO BMDCs (Figure 2G).A similar pattern of reactivity was observed when preQFLTg were cultured in thymic slices derived from WT, Qa1 b KO and b2mKO mice (Supplementary Figures 5B, C).This is consistent with the development of QFL T cells in Qa1 b KO mice, and suggests that the QFL TCR is cross-reactive with an alternative b2m-utilizing molecule.
To further explore the specificity of the QFL TCR for MHC 1 molecules, we used the DC-like cell line DC2.4 as a stimulator cell for preQFLTg thymocytes (Figure 2H; Supplementary Figure 5D).The response of preQFLTg thymocytes to DC2.4 cells was abolished by CRISPR/Cas9 mediated gene knock out of b2m and partially reduced by loss of Qa1 b , paralleling the results from stimulation with BMDC, and pointing to the recognition of an alternative MHC-1 ligand in this system (Figures 2G, H

QFL T cell selection by hematopoietic and non-hematopoietic cells
While conventional abT cells undergo positive selection by recognition of MHC molecules on thymic epithelial cells, MAIT cells and iNKT cells undergo selection via interactions with hematopoietic cells (4,5).To investigate the cell type requirements for selection of QFL T cells, we generated reciprocal bone marrow chimeric mice in which either the donor cells or the host cells are b2mKO, and therefore lack surface expression of Qa1, as well as the classical MHC I molecules H2-D, H2-K (Supplementary Figure 6).Interestingly, comparable numbers of QFL T cells were found in the thymus and spleen of the b2mKO>WT and WT>b2mKO chimeric mice (Supplementary Figures 6B, C), implying that QFL T cell development could occur efficiently on either non-hematopoietic or hematopoietic cells.To confirm these results, we generated reciprocal b2mKO chimeras using donor cells that expressed the QFL TCR transgene (Figure 3A).While there was some reduction in QFL T cell number in the thymus of b2mKO>WT compared to WT>b2mKO and wild type control chimeras (Figure 3B), similar numbers of QFL T cells were found in the spleen (Figure 3C).Thus, QFL T cell development is not strictly dependent on either non-hematopoietic or hematopoietic expression of MHC I.
We also examined whether QFL T cells that are selected exclusively by hematopoietic or non-hematopoietic cells retained their agonist selected phenotype.QFL thymocytes exhibited comparable CD8b downregulation but decreased PLZF expression when MHC I was restricted to non-hematopoietic cells (Figures 3D,  E; Supplementary Figure 6D).Similarly, expression of CD5 was decreased when MHC I was restricted to non-hematopoietic cells (Figure 3F; Supplementary Figure 6E).In the periphery, QFL T cells in chimeric mice that lacked MHC I on hematopoietic cells did not display elevated expression of CD44 (Figure 3G; Supplementary Figure 6F).Overall, the thymic phenotype of QFL T cells in b2mKO>WT chimeras is similar, but not identical, to that observed in Qa1 b KO mice (Figures 2D-F).These data suggest that Qa1 b on both hematopoietic and non-hematopoietic cells contribute to agonist selection, with hematopoietic cells playing the predominant role.

Impact of agonist selection on QFL T cell function
To test how agonist selection impacts the functionality of QFL T cells, we compared QFL T cells that arose in the presence or absence of Qa1 b for their ability to respond in-vitro to ERAAPKO splenocytes.QFL T cells from QFLTg showed extensive upregulation of activation markers and increased proliferation in response to stimulation compared to QFL T cells from QFLTg Qa1 b KO mice (Figures 4A, B).This implies that agonist selection on Qa1 b led to greater functional responsiveness, which could reflect greater functionality on a per cell basis, or an increased frequency of functional cells within the population.Since agonist selection partially correlates with selection on hematopoietic cells (Figure 3; Supplementary Figure 6), we also examined QFL splenocytes from reciprocal b2mKO and wild type bone marrow chimeric mice as a further test of the impact of agonist selection on function.QFL T cells from QFLTg>b2mKO mice (Hematopoietic cell (HC) selected) responded more robustly to ERAAPKO APCs compared to cells from QFLTg b2mKO>WT mice (non-HC selected) (Figures 4A,  B).Thus, QFL T cells that develop in the absence of Qa1 b , or in the absence of hematopoietically expressed MHC I, exhibit reduced functionality.

QFL thymocytes and splenocytes can populate the intestinal epithelial compartment
QFL thymocytes display an agonist selected phenotype that is enhanced by hematopoietic expression of MHC I. Given that agonist selection in the thymus can give rise to natural intraepithelial lymphocytes, we considered that QFL thymocytes might represent a population of IEL precursors.To test this idea, we injected Rag2KO neonatal mice with CD4-depleted QFL thymocytes from HC-only selected (QFLTg>b2mKO) or both HC and non-HC selected (QFLTg>WT) chimeric mice (Figure 4C).For comparison, we also injected Rag2KO neonatal mice with CD4depleted thymocytes or splenocytes from intact QFLTg mice.QFL T cells were found in similar numbers in the spleen and IEL compartment of the SI for all 4 donor populations (Figure 4D).Interestingly, IEL T cells derived from HC-only selected QFL thymocytes showed more pronounced downregulation of CD8b (~50%) when compared to IEL T cells derived from HC+ non-HC selected QFL T cells (~10%) (Figure 4E).Altogether, these data indicate that both QFL thymocytes and splenocytes contain IEL precursors, and that selection by hematopoietic cells favors CD8b downregulation in QFL IELs.

Discussion
Most studies of unconventional T cells have focused on 2 prominent populations, MAIT (MR1-restricted) and iNKT (CD1d restricted) cells, and much less is known about the development of T cells restricted to other MHC Ib molecules.Moreover, while it is known that non classical MHC molecules contribute substantially to the CD8aa natural IEL compartment (13, 15, 54), and there is evidence that thymic mature CD4-CD8-(DN) cells contain IEL precursors (21,22,25,59), it is unclear whether all natural IEL develop via a mature DN pathway.Here we have used both QFL TCR transgenic mice and FL9-Qa1 b tetramer staining of non-transgenic mice to investigate the development of a population of self-reactive Qa1 b restricted cells known as QFL T cells.QFL T cells are found in circulation as both naïve and memory phenotype CD8ab T cells, and in the IEL compartment as both CD8aa and CD8ab cells, whereas mature QFL thymocytes are predominantly CD8ab+CD4-and show signs of agonist selection.QFL T cells have a more relaxed requirement for positive selection compared to conventional CD8 T cells, requiring b2m on either hematopoietic or non-hematopoietic cells, but neither the restricting molecule Qa1 b , nor MHC Ia for positive selection.However, QFL thymocytes do require Qa1 b for agonist selection and full functionality.Our data highlight the promiscuous requirements for positive selection of a Qa1 restricted T cell population and identify an alternative CD8ab+CD4-pathway for development of CD8aa IELs.
The flexible thymic development of QFL T cells parallels their ability to give rise to T cells with both conventional and unconventional properties.Unconventional MAIT and iNKT cells require selection by their restricting MHC molecules on hematopoietic cells, giving rise to T cells that migrate directly to tissues and exhibit preformed effector program.On the other hand, conventional CD8 T cells require selection by their restricting MHC Ia molecules on thymic epithelial cells, producing circulating naïve T cells that lack effector programing.QFL T cells appear to have the option to develop by either of these pathways, with selection by Qa1 b on hematopoietic cells leading to a more unconventional phenotype, and selection via an alternative MHC I ligand giving rise to T cells that resemble conventional CD8 T cells.Interestingly, the ability to be selected on either hematopoietic or non-hematopoietic cells in the thymus has been reported both for another Qa1 restricted T cell population (9), as well as a T cell population restricted to the MHC Ib molecule H2-M3 (8).While the M3 restricted cells required M3 expression for thymic selection, hematopoietic selection led to T cells with more unconventional functional properties compared to nonhematopoietic selection.Thus, a flexible pattern of thymic selection leading to alternative functional programs may be a general feature of T cell reactive to Qa1 and H2-M3.
The IEL compartment harbors 2 distinct types of abTCR+CD8+ T cells: "induced" CD8ab T cells that are derived from conventional CD8 T cells following encounter with foreign antigen and differentiation into tissue resident memory T cells, and "natural" CD8aa IEL that are directed into an IEL program in the thymus due to their high self-reactivity (16,17).The observation that the same TCR clone can give rise to both types of IEL blurs the distinction between these two types of cells.Previous studies of natural IEL development have suggested a pathway in which some DP thymocytes that receive strong TCR signals escape clonal deletion by downregulating CD4 and CD8 to give rise to mature CD4-CD8-IEL precursors (IELp), that can eventually migrate to the gut and upregulate CD8aa (17,22).Our data are consistent with an alternative pathway for IEL development in which agonist selection leads to a mature CD8ab+CD4-thymic IELp.While the ability of CD4-depleted thymocytes to repopulate the IEL compartment of Rag2KO mice does not rule out a contribution from CD4-CD8aprecursors, the absence of detectable CD4-CD8a-QFL thymocytes in non-transgenic mice, as well as the agonist phenotype of thymic QFL CD8SP, strongly suggests that the CD4-CD8ab cells are the relevant thymic precursor population.Moreover, the partial downregulation of CD8b observed on mature QFL thymocytes and on some QFL IEL T cells, suggests that CD8b expression may be unstable in QFL cells, leading to partial or full downregulation once they arrive in the IEL compartment.The suggestion is also consistent with the observation that splenic QFL T cells, which are uniformly CD4-CD8ab, can give rise to IELs expressing intermediate levels of CD8b upon transfer into Rag2KO recipients.Altogether, these data are in line with earlier studies of thymocytes agonist selection in organ culture that also implicated mature CD8SP as a thymic precursor to natural IEL (49,50).A detailed understanding of the developmental pathways and signals involved in QFL IEL T cell development awaits further investigation.
Our data, together with published observations, support the notion that MHC-E restricted CD8 T cells are generally crossreactive.Using an in vitro stimulation assay with pre-selection QFL thymocytes, which reads out relatively weak TCR signals compatible with positive selection (57,58), we found that QFL thymocytes can respond to Qa1 b KO APCs, but not to APC lacking Qa1 b as well as both K b and D b classical MHC1a molecules.Thus, cross-reactivity to classical MHC-1 molecules may account for the positive selection of QFL thymocytes in the absence of Qa1 b .In addition, another Qa1 b restricted clone was shown to cross react with an MHC Ia molecule (60), although it was dependent on Qa1 b for its positive selection (9).The MHC-E restricted response to a CMV-vectored HIV vaccine showed extremely broad reactivity, with detectable responses to 4 epitopes for every 100 amino acids (61).In this regard, it is intriguing that QFL T cells show strong preferential use of Va3.2 (encoded by TRAV9N/D-3) (41).Va3.2 is preferentially used by CD8 T cells compared to CD4 T cells (62) and has been suggested to be inherently reactive to MHC I (63).In addition, Va3.2 is enriched in a subset of natural IELs (22), and is used by another Qa1 b -restricted CD8 T cell clone (9).Moreover, we found that the frequency of Va3.2+ CD8 T cells is substantially increased in K b D b KO mice (Supplementary Figure 4D).Altogether, these observations suggest that Va3.2 may work together with Qa1 b , and perhaps other non-classical MHC I molecules, to generate self-reactive T cells with a propensity to give rise to memory phenotype and natural IEL T cells.
If MHC-E reactive CD8 T cells are inherently cross-reactive, how do they escape negative selection in the thymus?While thymocyte intrinsic mechanisms, such as downregulation of CD4 and CD8 may contribute (56), it is interesting to consider how properties of the MHC molecules may also play a role.In particular, MHC-E molecules tend to be expressed at lower levels on the cell surface compared to MHC Ia molecules (9,64,65), a property that may be linked to their atypical peptide presentation pathway (66, 67) and/or low surface stability (64).In addition, MHC-E molecules predominantly express a single self-peptide derived from MHC Ia leader peptides (68,69), and may not present a large array of selfpeptides in healthy cells.Indeed, it has been proposed that MHC-E molecules may monitor alterations in the MHC Ia peptide presentation pathway that occur upon viral infection or cellular transformation (10,31,33), changes which may be mimicked by conditions of cellular stress.According to this notion, MHC-E restricted T cells may undergo rare or transient encounters with high affinity self-peptide-MHC-E complexes on stressed cells during their development in the thymus, allowing them to experience agonist selection signals while avoiding negative selection.

Generation of the QFLTg mouse
The TCR alpha and beta chain sequences from the QFL specific BEKo8z hybridoma (31,41) were cloned and amplified from the genomic DNA of the BeKoz Hybridoma.The TRAV9N/D-3 TCR alpha chain was cloned with the forward primer (5' AAAACCCGGGCCAAGGCTCAGCCATGCTCCTGG) with an added XmaI cutting site at 5' end of the DNA sequence and a reverse primer for TRAJ21 (5' AAAAGCGGCCGCATA CAACATTGGACAAGGATCCAAGCTAAAGAGAACTC) with an added Not1 cutting site at the 5' end of the DNA sequence.The TCR beta chain was cloned with the forward primer (5' AAAACTCGAGCCCGTCTGGAGCCTGATTCCA) with and added Xho1 cutting site at the 5' end of the DNA and a reverse primer for TRBJ2-7 (5' AAAACCGCGGGGGACCCAG GAATTTGGGTGGA) with a SacII cutting site flanking the 5' end of the DNA sequence.The cloned TCR alpha chain was cloned into pTa cassette vector by inserting it between the Xmal and Not1 sites, while the TCR beta chains were cloned into pTb cassette vector in between the Xhol and SacII sites (71).The ampicillin resistance gene was removed from pTa and pTb cassette by EarI enzyme digest.The QFL transgenic mice were generated on the B6 background in the Cancer Research Laboratory Gene Targeting Facility at UC Berkeley under standard procedures.The QFL mice were maintained on the B6 background and bred once with B6 5.1 mice to generate (QFLTgxB65.1/2)background mice for use in experiments.Founder mice were identified by flow cytometry and PCR genotyping of tail genomic DNA using primers mentioned above.
Generation of K b D b KO mice K b D b KO were generated by the Gene Targeting Facility at UC Berkeley using Cas9/CRISPR-mediated gene targeting.The H-2K1 gene was targeted using an sgRNA (5' GTACATGGA AGTCGGCTACG 3') that aligned with the sense strand and the H-2D1 gene was targeted using an sgRNA (5' AGATGTACCG GGGCTCCTCG 3') that aligned with the antisense strand.Wildtype C57BL/6J mice were originally obtained from the Jackson Laboratories.Zygotes were obtained from super ovulated C57BL/6J females for CRISPR/Cas9 targeting knockout experiment.In brief, CRISPR mix (i.e., Cas9 protein and sgRNAs) was introduced to zygotes by electroporation as previously described (72).The embryos were then transferred to 0.5dpc pseudo pregnant females (CD-1, Charles River Laboratories) with oviduct transfer.When the pups were born, tails samples were collected for DNA extraction and genotyping.The resulting founder mice were identified by flow cytometry.The H-2K1 gene had a 2bp deletion (5' where the sgRNA targeted.The H-2D1 gene had a 15bp deletion where the sgRNA targeted (5

Preparation of cell suspension
Thymi, and spleens were mechanically dissociated in FACS buffer (0.5% BSA in PBS) or complete RPMI (10% FBS) to generate single-cell suspensions that were then passed through a 70mm filter.Intraepithelial lymphocytes (IELs) were isolated from the small intestine as previously described (73).Briefly, small intestine was cut to 1cm pieces and washed with cold CMF.Tissue pieces were allowed to settle and CMF was poured off.The tissue was then digested with DTE solution for 30 min at 37C in a 50mL conical tube.Tissue pieces were centrifuged at 1,500rpm for 5 min at 4C. Supernatant was collected and centrifuged at 1,500rpm for 5min at 4C. Lymphocytes were isolated by percoll separation utilizing 40% and 80% percoll (22).The percoll solution was centrifuged at 2000rpm with no brake for 20 min at room temperature.Lymphocyte layer was then washed with PBS and collected.Splenocytes were then RBC lysed using ACK lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2EDTA) for 5 minutes at room temperature.

Tetramer enrichment
Single-cell suspensions of thymi and spleens were generated as described above.Cells were incubated in 50nM Datsatinib (Sigma Aldrich, CDS023389-25MG) for 30 minutes at 37°C and then stained with tetramer in 2.4G2 supernatant for 1 hour at room temperature.After staining, cells were washed and incubated with Anti-PE MicroBeads (Miltenyi Biotec, #130-048-801) in MACS buffer (0.5% BSA) for 30 minutes at 4°C.Cells were then positively enriched for tetramer+ T cells using a magnetic column (Miltenyi Biotec.)according to manufacturer's instructions and washed before extracellular staining.

Bone marrow dendritic cell culture in vitro stimulation
Bone marrow cells were harvested as described above and RBC lysed using ACK lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2EDTA) for 5 minutes at room temperature.Bone marrow cells were resuspended in cRPMI and seeded at 5 x 10 6 cells per milliliter in 24 well plates.Cells were supplemented with GM-CSF (Peprotech, #315-03-20UG) until day 4 and adhering cells were harvested on day 6 using EDTA.6 x 10 5 CD11c+MHC-II+ bone marrow cells per milliliter were seeded in 24 well plates.Preselection QFL thymocytes were generated by crossing QFL TCR transgenic mice onto a non-selecting, MHC-I deficient background (b2M-/-).Thymic single-cell suspensions were generated as described above.Thymocytes were resuspended in cRPMI and seeded at 4 x 10 6 cells per milliliter.
In vitro stimulation with DC2.No:3548) at 3x10 5 cells per well.Cells were left to settle for 1 hour at 37°C 5% CO 2 .DC2.4 cells were then treated with 5ng/ml Recombinant Mouse IFN-g (Biolegend, Cat.No:575302) for 24 hours, then Recombinant Mouse IFN-g was washed off.Singlecell suspensions of QFLTgb2mKO thymi were prepared as described above.Thymocytes were overlaid at 1x10 5 cells per well and cultured at 37°C 5% CO 2 for 24 hours, then harvested for flow cytometric analyses.

Thymic tissue slice cultures
Thymic lobes from wild type, Qa1 b KO and b2mKO mice were gently isolated and any connective tissue was removed.Lobes were embedded into 4% agarose with a low melting point (GTG-NuSieve Agarose, Lonza) and sectioned into 400-500mm slices using a vibratome (VT1000S, Leica).Thymic slices were overlaid onto 0.4mm transwell inserts (Corning, Cat.No.: 353090) in 6 well tissue culture plates with enough cRPMI under the insert to reach the slices.Pre-selection QFL (2.5x10 5 ) thymocytes were overlaid onto each slice and allowed to migrate for 3 hours, after which excess thymocytes were removed by gently washing with PBS.Slices were cultured at 37°C 5% CO2 until harvested for analysis.For flow cytometry, thymic slices were mechanically dissociated into singlecell suspensions prior to staining.

Bone marrow chimeras
Host mice were depleted of NK cells by I.P. injecting anti-NK1.1 (PK136, Leinco Technologies, #N123) at 100ug/100uL every 24Hrs for two days, for a total of 200ug of depleting antibody.Mice were irradiated in two doses of 600 rads (total of 1,200rads), with a resting period of 16hrs between doses.Mice were maintained on antibiotic water (Trimethoprim/Sulfamethoxazole) 4 weeks following irradiation.Bone marrow was harvested from the femur of donor mice using standard techniques.Red blood cells were lysed using ACK lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2EDTA) for 5 minutes at room temperature.Cells were depleted of CD4+ T cells by staining with CD4 PE-conjugated antibody (RM4-4) for 20 minutes at 4°C and then with Anti-PE MicroBeads (Miltenyi Biotec.) as described above.The labeled cells were washed, resuspended in MACS buffer, and then passed through a magnetic column (Miltenyi Biotec.).Flow-through (CD4-depleted bone marrow cells) was washed, resuspended at (4x10 6 cells) in 100uL of PBS and i.v.injected into recipient mice.Bone marrow chimeras were analyzed 8-11 weeks following reconstitution.

CFSE labeling
Cells were resuspended in 5uM CFSE proliferation dye (ThermoFisher #C34554) and incubated at 37°C for 9 minutes.Cells were then washed by addition of pre-warmed cRPMI while vortexing.Cells were then washed again with pre-warmed PBS and resuspended at the desired concentration.

In vitro stimulation with splenocytes
Single cell-suspensions of splenocytes were generated and RBC lysed as described above.Antigen presenting cells (APCs) were prepared by depleting splenocytes of CD4, CD8, and NK1.1 expressing cells using a magnetic column as described above.APCs were seeded at 4x10 5 cells per well in a 48 well plates.Responding cells were splenocytes from QFLTg mice that were depleted of CD4, NK1.1, B220, and CD19 expressing cells using a magnetic column as described above.Cells were then CFSE (ThermoFisher #C34554) labeled as described above and seeded at 1x10 5 cells per well.
).Interestingly, triple KO of Qa1 b , K b , and D b in DC2.4 cells reduced activation close to background levels, whereas double knock out of Qa1 b with either K b , or D b led to stimulation that was intermediate between the triple KO and Qa1 b KO cell lines.Together these data suggest that classical MHC-1 molecules may contribute to the positive selection of QFL T cells in Qa1 b KO mice.
4 cells WT, Qa1 b KO, Qa1 b K b D b KO, Qa1 b K b KO, Qa1 b D b KO, and b2mKO DC2.4 cells were plated in 48 Well Cell Culture Plates (Corning, Cat.